Transplantation of organs, tissue or cells from one genetically distinct person (donor) to another (recipient) is hindered by the recipient's immunologic rejection of the donated organs or cells. This rejection phenomenon is understood to involve both cellular and humoral mechanisms, mediated respectively by T cells and antibodies. The recipient's immune system targets distinguishing histocompatibility antigens on the transplanted cells. Except in rare cases, the donor's histocompatibility antigens will not match exactly the recipient's histocompatibility antigens, and the recipient's immune system attacks the incompatible donor organs or cells.
With respect to immunologically mediated rejection, the most potent of the histocompatibility antigens are the major histocompatibility complexes (MHC) known as the human leukocyte antigens, HLA-A, HLA-B and HLA-C. Although originally defined by their presence on the cell membranes of human leukocytes, they have long been recognized to be present on virtually all of the nucleated cells of the human body. Since each person receives genes encoding one set of these antigens from each parent, human cells typically express six major HLA antigens. In addition to the major histocompatibility antigens, there are several minor histocompatibility antigens.
When tissue or cells are transplanted, it is desirable to match, to the maximum extent possible, the histocompatibility antigens of the donor and the recipient. The best immunologic match between donor and recipient is between identical twins, since they share the same six major HLA antigens. In addition, identical twins also share the same minor histocompatibility antigens, and therefore organs or cells transplanted from one identical twin to the other are immunologically tolerated. In the far more common situation in which the donor and recipient are not genetically identical, some level of immunologic rejection of transplanted tissue regularly occurs. To minimize this rejection and permit survival of the engrafted tissue, efforts are routinely made to find the best match between donor and recipient. If an identical twin is not available, the next best choice is typically a non-identical sibling of the recipient sharing the same six major HLA antigens, a situation which occurs on the average in one out of four siblings. Such a six out of six HLA match between siblings is preferable to a six out of six match between unrelated individuals, because the matched siblings will also more likely share at least some minor histocompatibility antigens inherited from their common parents. Yet, because they are not identical siblings, there is a high probability of some difference in the minor histocompatibility antigens, and the donor and recipient will almost certainly be sufficiently distinct in terms of cellular antigens that some level of rejection will occur following transplantation of tissue from one sibling to another.
The adverse reactions following transplantation of an organ or tissue from one genetically distinct individual to another can be profoundly dangerous. The primary adverse reaction is immunologic rejection of the transplanted organ or tissue. If the organ is life-sustaining, such as a heart, liver or lung, the destruction of that organ may lead directly to the death of the patient. In other circumstances, such as rejection of insulin producing pancreatic islet cells or kidneys, the quality of life of the recipient may be devastated by the tissue rejection. In order to prevent or limit the rejection, patients typically receive a combination of immunosuppressive drugs, which introduce their own major side effects. These drugs are usually globally immunosuppressive, thereby greatly increasing the susceptibility of the recipient to serious infections, often by organisms against which an uncompromised immune system would readily defend. The individual immunosuppressive drugs each have their own set of other adverse effects, especially when used in the dosages necessary to inhibit rejection of transplanted organs. For example, high doses of prednisone precipitate diabetes mellitus and hypertension, while simultaneously causing demineralization of supporting bones. Another commonly used immunosuppressive drug, cyclosporine A, has major toxic effects on the kidney. Globally immunosuppressive treatments also increase the susceptibility of transplant recipients to opportunistic infections, against which normal individuals have strong defenses.
These adverse effects have stimulated searches for therapies that can more selectively suppress the rejection of transplanted tissue, while leaving the remainder of the immune system intact and not injuring other important organs. An especially promising approach has been the use of a conventional Photopheresis device to deliver the immunotherapy generally referred to herein as “Transimmunization” to prevent or reverse rejection of transplanted organs. Depending on the circumstances, the therapeutic impact of the Transimmunization can be enhanced by following the conventional Photopheresis step with an overnight incubation phase, prior to returning the treated cells to the patient. Transimmunization may be accomplished using a Photopheresis apparatus, although Transimmunization may also be accomplished without the use of a Photopheresis apparatus, using other methodology.
A controlled trial comparing conventional Photopheresis plus conventional immunosuppression with conventional immunosuppression alone in the prevention of rejection of transplanted hearts was reported by Barr et al., Photopheresis for the prevention of rejection in cardiac transplantation, New England Journal of Medicine, Vol. 339, No. 4, 1744-51, Dec. 10, 1998. That study revealed that the addition of Photopheresis to the conventional immunosuppressive regimen quite significantly and safely reduced the number of rejection episodes, thereby markedly diminishing the need for dangerous boosting of the levels of toxic conventional immunosuppressive drugs. Similarly, in Greinix et al., Successful use of extracorporeal photochemotherapy in the treatment of severe acute and chronic graft-versus-host disease, Blood, Vol. 92, No. 9, 3098-3104, 1998, and in Greinix et al., Extracorporeal photochemotherapy in the treatment of severe steroid-refractory acute graft-versus-host disease: a pilot study, Blood, Vol. 96, No. 7, 2426-31, 2000, the authors describe testing which revealed that Photopheresis was particularly effective in reversing the adverse effects (known as graft-versus-host-disease or GVHD) following transplantation of bone marrow or stem cells.
One mechanism that is involved in the efficacy of Photopheresis has been recently deciphered. The flat plastic ultraviolet exposure system, a component of the Photopheresis apparatus, can cause the transformation of blood monocytes to dendritic antigen presenting cells (dendritic cells) as a result of the forces imposed on the monocytes as they flow past the plastic surface in a conventional Photopheresis apparatus. Since the therapeutic benefits resulting from the use of these dendritic cells are caused by the transfer of tissue antigens to dendritic cells capable of immunization of the patient against these antigens, the immunotherapy is referred to as “Transimmunization.” Therefore, Transimmunization is a treatment that can, in one embodiment, be accomplished with a Photopheresis apparatus. Alternatively, the Transimmunization treatment may be performed using any other appropriate device having plastic channels which can induce differentiation of monocytes into dendritic cells. One important difference between the Transimmunization process described herein and conventional Photopheresis is the recognition that the necessary tissue antigens can best be delivered to the new dendritic cells by overnight ex vivo incubation, prior to return to the patient of the antigen loaded dendritic cells.
In Photopheresis, a photoactivatable agent, such as 8-methoxypsoralen (8-MOP), is activated by exposure to ultraviolet A (UVA) in extracorporeally circulated blood, causing the 8-MOP to form photo adducts with pyrimidine bases of DNA and tyrosine containing cytoplasmic proteins. The positive clinical sequelae caused by Photopheresis result from the patient's immunologic response to the reinfused treated blood. The resulting immune response can, in the best responders, lead to the selective suppression or even elimination of the pathogenic clone(s).
As stated above, it has more recently been discovered that the passage of the blood through the plastic ultraviolet exposure chamber of the Photopheresis device can stimulate the conversion of blood monocytes to dendritic antigen presenting cells (DC), the most potent initiators of cellular immune reactions. Two phenomena contribute to the clinical success of Transimmunization. In certain situations, it is desirable to produce a positive immunologic reaction or immunization against disease causing cells which are distinguished from their normal counterparts by their surface display of distinctive molecular targets which can be targeted immunologically. One such situation is cancer, in which the malignant cells display distinctive tumor antigens. In other situations, it is desirable to suppress an immunologic reaction which actually causes the disease. Those situations include immunologic rejection of a transplanted organ or graft-versus-lost disease (GVHD), in which stem cell preparations transplanted from a donor to a recipient include donor T cells which then recognize the recipient's tissues as “foreign”, proliferate to form many copies of themselves in the recipient and dangerously attack the normal cells of the recipient.
In the cancer situation, the malignant cells may be damaged so that they will be ingested by the newly formed DC, which are then permitted to develop into mature DC which subsequently present the distinctive antigens of the pathogenic cells to a responding immune system, thereby generating a “positive” immunologic reaction capable of inhibiting or destroying a cancer. The CD8 (and probably also CD4) T cell responses caused or enhanced by this treatment can often be sustained for long periods of time. As described in U.S. patent application Ser. No. 09/928,855, now U.S. Pat. No. 6,607,722, the process can be refined to maximize ingestion of apoptotic pathogenic T cells by newly formed DC and thereby maximize the strength of the anti-cancer immunologic reactions generated.
However, a variation of the same procedure can also be used to selectively suppress undesirable immunologic reactions, such as organ transplant rejection or GVHD, through the suppressive actions of DC that have remained immature. Unlike mature DC, immature DC transmit specific suppressive signals to T cells, because the immature DC have not yet developed the arsenal of membrane molecules capable of stimulating strong positive immunologic responses. For example, immature DC are deficient in costimulatory molecules, such as B7.1 and B7.2, which can contribute to the delivery of positive signals to responsive T cells. Instead of causing positive, or immunizing responses, these immature DC can selectively suppress reactions against precisely those antigens which they have ingested, processed and presented on their surface. Studies in an experimental model of conventional Photopheresis revealed the capacity of that treatment to selectively suppress rejection of transplanted tissue. Specifically, when skin was transplanted from a donor black mouse to a genetically completely distinct white mouse, the transplanted skin was completely rejected within 14 days. This was anticipated, since the donor and recipient mice differed in terms of histocompatibility antigens to a level equivalent to a six out six mismatch in humans and since skin is the most immunogenic solid organ. Following the rejection of the transplanted skin, the recipient mouse was sacrificed and its spleen, containing markedly expanded clones of those T cells causing the rejection, as well as tissue monocytes, were brought into single cell suspension. Then, in a system devised to mimic conventional photopheresis, the suspended T cells were exposed in a petri dish to UVA activated 8-MOP and then returned intravenously to a mouse genetically identical to the original recipient, thereby immunizing this new mouse against the clones of T cells involved in the rejection of the transplanted skin.
This new mouse then received new skin transplants: one from the same original donor strain and another from a third mouse strain completely unrelated to either of the other two strains. Instead of being rejected within 14 days as before, the transplanted skin from the original donor strain now survived intact for the full 42 days of the experiment. In contrast, the simultaneously transplanted skin from the third unrelated strain was rejected within the 14 days. The selective suppression of the rejection of the skin graft could be transferred to another set of mice, genetically identical to the original recipient, by transfusion of recipient T cells. These results demonstrated that the experimental model of Photopheresis led to donor specific suppression of the rejection of the transplanted skin and that this suppression was mediated by selectively suppressive T cells induced by the procedure. These tests are reported in more detail in Yamane et al., Suppression of anti-skin-allograft response by photodamaged effector cells—the modulating effects of prednisone and cyclophophamide, Transplantation, Vol. 54, 119-124, No. 1, July 1992; Perez et al., Induction of a cell-transferable suppression of alloreactivity by photodamaged lymphocytes, Transplantation, Vol. 54, 896-903, No. 5, November 1992; Perez et al., DNA associated with the cell membrane is involved in the inhibition of the skin rejection response induced by infusions of photodamaged alloreactive cells that mediate rejection of skin allograft, Photochemistry and Photobiology, Vol. 55, 839-849, No. 6, 1992.
Paradoxically, when the experiment was altered so that the donor strain differed from the recipient strain by only minor histocompatibility antigens, the transplanted skin could be kept intact on the recipient for only 21 days. This was longer than in untreated controls, but only half as long as when skin from the completely unrelated strain was transplanted to prepared recipients. Although puzzling at the time, it appears that the stronger the reaction that is being suppressed, the more effective it is. This is probably due to the preferential sensitivity of high affinity T cells, more readily generated by potent immune reactions, to be suppressed directly by the immature DC produced in the experimental Photopheresis procedure. Importantly, this finding suggests that the Transimmunization process described below may be most effective in preventing rejection of transplanted organs when the donor and recipient are mismatched by one or more HLA antigens. Accordingly, Transimmunization may dramatically augment the donor pool of transplantable tissue. The modification of Transimmunization which is described in this application will further augment the donor pool of transplantable tissue, by enhancing the suppressive effects of Transimmunization.
Methods for suppressing the immune response of individuals to transplanted tissue or cells using the Transimmunization procedure in combination with skin grafts between transplant recipients and donors have been described previously in pending U.S. patent application Ser. No. 10/217,856. The present invention relates to methods of suppressing the immune response of individuals to transplanted tissue or cells using a modified Transimmunization procedure to induce formation of dendritic cells, and truncating the maturation of the dendritic cells at a stage where they will inactivate selected T cells which would otherwise participate in GVHD or in rejection of a transplanted organ or tissue.
Immature dendritic cells may be used to suppress undesired immune system responses in individuals receiving transplanted bone marrow, stem cells, organs or tissues. Immature dendritic cells can preferentially suppress immune responses due to their relative deficiency in costimulatory molecules, such as CD-80 and CD-86 (also known as B7.1 and B7.2 respectively). In order for an antigen presenting dendritic cell to stimulate positive immune responses from antigen-specific T cells, two signals must be transmitted from the antigen presenting dendritic cell to the antigen-specific T cell. A first signal is transmitted from the antigen presented on the surface of the dendritic cell to the T cell via the binding of the antigen with a complementary surface T cell receptor. The second signal is transmitted by costimulatory molecules on the surface of the dendritic cell. If the antigen signal is transmitted in the absence of the costimulatory signal, the antigen-specific T cell is inactivated or eliminated, rather than being stimulated to produce a positive immunologic response to cells displaying the transmitted antigens. It is known that immature dendritic cells are deficient in costimulatory molecules, and therefore transmit negative or suppressive signals to those T cells having receptors for the presented antigens. Accordingly, dendritic cells may be produced having a truncated maturation, and the immature dendritic cells can be used to suppress the response of antigen specific T cells in transplant recipients.
Cancer patients are often prepared for bone marrow/stem cell transplants from genetically distinct individuals by first receiving large doses of chemotherapy to accomplish two goals: diminution of the tumor burden and weakening of the immune system so that the transplanted cells will not be quickly rejected. This level of preparation is itself life threatening, since the cancer patient's own bone marrow is largely destroyed by the preparative chemotherapy. If the transplanted cells do not take and ultimately reconstitute the patient's bone marrow, the patient will succumb to infections, anemia, hemorrhage, etc. When the bone marrow/stem cell transplant does successfully reconstitute the patient's immune system, that immune system is repopulated by the cells of the donor. These donor cells then recognize the recipient's tissue as foreign and attack (reject) the recipient's own organs in a process called graft versus host disease (GVHD). Most prominently attacked in this resulting GVHD are the skin (which can slough), the liver (which can fail) and the intestinal tract (which can cease to function properly and hemorrhage). Life-saving reversal or suppression of GVHD is quite difficult with conventional treatments, which are usually quite toxic.
Remarkably, in recent years, it has become clear that a controllable level of GVHD may be of great benefit to the cancer patient. Since the donor cells react against recipient histocompatibility antigens, and since the residual cancer cells are also recipient cells bearing the patient's histocompatibility antigens, a certain level of “graft-versus-tumor” reaction or GVTR commonly accompanies the undesirable other components of GVHD. Those cancer patients who survive GVHD following bone marrow/stem cell transplants appear to have an improved survival from their cancer, since recurrences are less frequent. Therefore, a fine line exists between the toxic effects of GVHD and the beneficial ones of GVTR. In an ideal situation, a treatment could suppress GVHD while leaving a partial GVTR, directed at those weaker antigens which distinguish the malignant cells from the benign cells of the recipient. The methods of the present invention may be used to inactivate donor T cells which would otherwise attack the recipient's healthy tissues, while leaving intact T cells which attack tumor cells displaying a different set of antigens.
In the organ transplantation situation, the recipient's T cells attack the transplanted organ, as the cells of the transplanted organs display antigens that are not identical to the antigens present in the recipient's cells. It would be desirable to find a treatment that could inactivate the recipient's T cells from attacking cells displaying the donor's antigens, while not effecting the ability of the recipient's T cells from recognizing and attacking infectious microbes or other disease causing agents.